Although there has been extensive work developing microfabrication and microfluidic technology for biomedical applications, there has still been limited progress in moving the technology broadly into biomedical research laboratories. Part of the issue is a lack of familiarity with the capabilities of microfabrication on the part of biomedical researchers. In addition, many potential research projects have constraints that require extensive customization and multiple design iterations, which may not be achievable with the limited number of commercial products available. In an effort to lower the barriers for applying microfabrication techniques to a wide range of biomedical problems, over the past year we have developed an in-house microfabrication capability which enables us to make single-layer templates for PDMS using a convenient dry-film resist process. Although the resolution, device yield, and complexity are considerably lower than those achievable with a dedicated cleanroom, they are nonetheless sufficient for many experiments on cells. Furthermore, the instrumentation complexity, fabrication cost, and turnaround time are greatly reduced, enabling rapid cycling through design parameters as needed. This year, considerable effort was spent on developing and optimizing protocols for patterning two commercial dry-film resists and SU-8, a spin-on resist. As a result, we are able to reliably pattern features with lateral dimensions as small as 15 microns, and with heights ranging from 15 microns to a few hundred microns. In addition, we developed a capability for maskless photolithography using a digital light projector;using this instrumentation we can make a microfluidic template from a bitmap image in a few hours or less. Finally, we also have developed protocols for surface modification of PDMS and other polymers, including the irreversible bonding of PDMS to glass using instrumentation in our laboratory, as well as techniques for connecting devices to flow-control instruments, such as syringe pumps. Through continued collaboration with scientists at NIST, we are also able to access the nanofabrication facilities at NIST to make more complex and higher-tolerance structures as needed. These new capabilities have found application in several initial projects during this past year, all of which are ongoing. The first is the fabrication of agarose microfluidic devices, on a platform compatible with high-resolution fluorescence and two-photon imaging, for the study of chemotaxis in a 3-D collagen matrix. The second project is an effort to confine B-T cell pairs in PDMS microwells such that the intercell junction is perpendicular to the optical axis, in order to enable high-speed high-resolution imaging of the synapse formation. A third project is the development of gratings for phase-contrast x-ray imaging, for which we are using both the NIST nanofabrication facilities as well as our own equipment. In addition, we have been using our capability to spin-coat thin polymer films to make thin poly(ethylene co-vinyl acetate) (EVA) films for use in operator-independent, high-resolution, laser capture microdissection. This work is part of an inter-institute Director's Challenge project with researchers in NIMH, NCI, and NICHD, aimed at developing methods for tissue-based capture of subcellular structures for mass spectrometry-based proteomic analysis. To this end, we have developed protocols for making capture films from several different grades of EVA with thicknesses ranging from 0.5 microns to a few microns, on flexible and rigid substrates. Finally, we continue work on a project aimed at miniaturizing the LIPS assay, which uses a fusion protein consisting of Renilla luciferase and an antigen of interest to probe for antibodies in human serum. In its current format, the assay is performed in a 96-well filter plate, and has been shown effective in detecting a number of autoimmune conditions and infectious diseases. Moving the assay to a microfluidic format could significantly speed the analysis, permit multiplexing, and also facilitate the application of this assay to point-of-care diagnostics. Previous experiments using cell extracts containing the fusion protein and commercial anti-CFLAG antibodies in lieu of serum had shown that the positive signal levels are acceptably high even in the miniaturized format. This year, our efforts focused on increasing measurement reproducibility and verifying function of the miniaturized assay with a panel of serum samples.